Biomining is, in general terms, the use of microorganisms for metal recovery from mineral ores. Its most traditional expression is bioleaching, but not only this process is understood as biomining, but also the monitoring and intervention in such process, as these techniques are complex and are under constant development; and also laboratory research associated to process improvement or the development of new methodologies.
Until now, bioleaching continues to be the most important process in biomining field, and is defined as a method to solubilize metals from complex matrixes in an acid medium, using direct or indirect microorganism action. The microorganisms that are useful in these processes belong to Bacteria or Archaea kingdoms, and fulfill two basic conditions: they are acidophilic and chemolithotrophic.
Microbiological Diversity in Communities Associated to Bioleaching Processes.
Various microorganisms have been described to be useful in bioleaching processes, and ten taxons could be identified among them: 3 genera and 2 species from the Bacteria kingdom, namely Acidiphilium sp., Leptospirillum sp., Sulfobacillus sp. genera and Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans species, and five genera from the Archaea kingdom, namely Acidianus sp., Ferroplasma sp., Metallosphaera sp., Sulfolobus sp. and Thermoplasma sp. (Rawlings D E. Heavy metal mining using microbes. Annu Rev Microbiol. 2002; 56:65-91; Rawlings D E. Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates. Microb Cell Fact. 2005 May 6; 4(1):13).
Factors Determining Diversity and Metabolic Activity of the Microbial Community Associated to a Bioleaching Process.
Each of the above mentioned genera or species catalyzes different reactions and require in its turn different conditions to perform such reaction, which could be, for instance, aerobic or anaerobic, or could require some specific nutrient. Therefore, the environmental conditions in which a bioleaching process is performed will modify the bacterial composition of the community.
Additionally, the participation of microorganisms in a bioleaching process has been proposed to be direct and/or indirect (Rawlings D E. Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates. Microb Cell Fact. 2005 May 6; 4(1):13). When the action is direct, microorganisms directly oxidize the target metal or its counter-ion, in both cases liberating into the solution a target metal ion. On the other hand, when the action is indirect, the substrate of the microorganism is not the target metal neither its counter-ion, but instead chemical conditions are generated that allow the solubilization of said metal, either by acidification of the medium (e.g., by generating sulfuric acid) or by the generation of an oxidizing agent that ultimately interacts with the salt (metal and counter-ion) to be solubilized.
Regarding this aspect, it is possible that the bacterial community changes its species composition as a function of the bioleaching type being performed in different mineral samples and/or the environmental conditions in which this process is carried out.
For instance, Acidithiobacillus species are able to catalyze the oxidation of reduced sulfur compounds (e.g., sulfide, elemental sulfur, thionates, etc.) using oxygen as electronic acceptor and generating sulfuric acid as final product and reducing species like sulfite and thiosulfate as intermediate products, which allows the solubilization of metals associated to sulfides in the mineral. Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans are able to catalyze the oxidation of iron(II) to iron(III) using oxygen as electron acceptor, being the generated iron(III) a great oxidizing agent that can oxidize sulfides in the mineral or any other compound to be oxidized.
The usual mining practice in bioleaching processes is to leave a mineral heap in an acid medium, generally sulfuric acid, and constantly remove the acid medium to recover the metal by electrolysis. Usually heaps in which the recovery yield of the metal is efficient are obtained, and also “inefficient” heaps that have a low yield under the same operation conditions and characteristics of the substrate to be leached. The explanation to this unequal result requires the elucidation of differences in abundance and types of species in the microbiological community between both heaps. In this way, the low yield problem could be explained by the microbial community composition, and could be solved in its turn by inoculation of microorganisms that catalyze the reaction to be maintained during the bioleaching process. However, a method that enables to quantify the population of archaea and bacteria useful in biomining processes is not available up to this date.
In this patent, a method is described that solves the technical problem previously described, by designing a method to identify and quantify the presence of known microorganisms that are most relevant in biomining processes, namely the bacteria: Acidiphilium sp., Leptospirillum sp., Sulfobacillus sp., Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans; and the archaea: Acidianus sp., Ferroplasma sp., Metallosphaera sp., Sulfolobus sp. and Thermoplasma sp.
Nested polymerase chain reaction (PCR) was the technique selected to develop this method. In this technique, a conserved genome region of the microorganisms is firstly amplified in a first PCR reaction, either on bacteria or archaea. We have selected gene 16SrDNA as the conserved region. Then, taxon-specific primers (targeting genera or species) are used to identify the presence of target microorganisms in a second PCR reaction. This second PCR reaction is performed using an equipment that allows measuring the increase of amplified product in each amplification cycle, and this information allows the quantification, by interpolation, of the original abundance of the target genome in the sample being analyzed. PCR reaction under these conditions is called quantitative PCR or qPCR.
A critical step in nested PCR technique is the design of primers for the second amplification reaction, which have to be specific for the taxon to be determined, and this aspect has a vital importance in this particular case, as the samples to which the process will be applied will usually be metagenomic samples. Therefore, it is necessary to reduce the possibility of primer unspecific hybridization to sequences present in the genome of microorganisms that have not yet been identified in the community. We have generated two fundamental tools for the design of these primers: firstly, a depurated 16SrDNA sequence database obtained from all disclosed 16SrDNA sequences; and a computational program for primer design that uses as input such database and allows designing thermodynamically stable taxon specific primers.
In the state of the art there are many examples of the application of nested PCR or qPCR, but none of them is focused to bacteria or archaea useful in biomining processes. For instance, J. L. M. Rodrigues et al (Journal of Microbiological Methods 51 (2002) 181-189) describe a qPCR to detect and quantify PCB-degrading Rhodococcus present in soil, where the 16SrDNA gene belonging to the strain with the target activity is sequenced, specific primers for said sequence are designed and qPCR reactions are carried out using said primers. In this document, a direct qPCR approach is used, instead of a nested qPCR, and it is directed to other type of microorganisms, whose handling has been widely studied and many techniques for DNA extraction are available. Another document that uses a similar approach is Patent Application EP 1 484 416, which discloses a method for the detection and quantification of pathogen bacteria and fungi present in an environment sample using qPCR. The method comprises the extraction of DNA from bacteria and fungi present in an environment sample, obtaining specific sense and antisense primers for each of the taxons to be detected and quantified; and performing qPCR reactions using a pair of primers for each of the target pathogens.
Although it is possible to enumerate documents in which microorganisms are identified and quantified using quantitative PCR techniques, as they are well known techniques in the art, the relevant point is the generation of a depurated database that allows to design specific primers and has not been implemented before for the identification of microorganisms useful in biomining processes, which is subject matter of this invention.